Assembly of Natively Synthesized Dual Chromophores Into Functional Actinorhodopsin

Abstract

Microbial rhodopsin is a simple solar energy-capturing molecule compared to the complex photosynthesis apparatus. Light-driven proton pumping across the cell membrane is a crucial mechanism underlying microbial energy production. Actinobacteria is one of the highly abundant bacterial phyla in freshwater habitats, and members of this lineage are considered to boost heterotrophic growth via phototrophy, as indicated by the presence of actino-opsin (ActR) genes in their genome. However, it is difficult to validate their function under laboratory settings because Actinobacteria are not consistently cultivable. Based on the published genome sequence of Candidatus aquiluna sp. strain IMCC13023, actinorhodopsin from the strain (ActR-13023) was isolated and characterized in this study. Notably, ActR-13023 assembled with natively synthesized carotenoid/retinal (used as a dual chromophore) and functioned as a light-driven outward proton pump. The ActR-13023 gene and putative genes involved in the chromophore (retinal/carotenoid) biosynthetic pathway were detected in the genome, indicating the functional expression ActR-13023 under natural conditions for the utilization of solar energy for proton translocation. Heterologous expressed ActR-13023 exhibited maximum absorption at 565 nm with practical proton pumping ability. Purified ActR-13023 could be reconstituted with actinobacterial carotenoids for additional light-harvesting. The existence of actinorhodopsin and its chromophore synthesis machinery in Actinobacteria indicates the inherent photo-energy conversion function of this microorganism. The assembly of ActR-13023 to its synthesized chromophores validated the microbial community's importance in the energy cycle.

Keywords: actinobacteria; carotenoid; dual chromophore actinorhodopsin; light-driven proton pumps; microbial rhodopsin; retinal.

Figure 1

Features of ActR-13023 vs. other microbial rhodopsins. (A) Multiple sequence alignment of ActR-13023 with bacteriorhodopsin (BR), proteorhodopsin (PR), xanthorhodopsin (XR), and actinorhodopsin from acI actinobacteria clades A, B, and C (ActR a–c). Gray highlights and the alphabet represent the helical segment. Cys/Thr on helix A and G (disulfide bond in ActR) is marked in yellow, DTE motifs in red, color tuning residue in green, phylogenetically differentiated ActR XR, Pro in blue, carotenoid interaction residues in cyan, and retinal-bound Lys in pink. (B) Functional visualization of ActR-13023 compared to light-driven outward proton pump. (C) Carotenoid interaction potential of ActR-13023. (D) Phylogenetic tree of ActR-13023 vs. other microbial rhodopsins (the tree was generated using MEGA X 10.1).

Figure 2

Characterization of ActR-13023 in Candidatus aquiluna. (A) C. aquiluna whole-cell spectrum (black) and expected ActR-13023 peak in the native cell (red). (B) Chromophore removal from C. aquiluna membrane and reconstitution with all-trans-retinal (1) membrane fraction with chromophore removed, (2) 1 h, (3) 24 h, and (4) 48 h after reconstitution, (5) carotenoid isolated from C. aquiluna. (C) Measurement of proton pumping potential of C. aquiluna.

Figure 3

Characterization of heterologously expressed ActR-13023. (A) Expression with additional all-trans-retinal (red) and bio-synthesized retinal (black). (B) Retinol conversion from β-carotene using the enzyme encoded by the C. aquiluna blh gene. (C) A light-induced differential spectrum of purified ActR-13023. (D) Flash-induced transient absorbance changes during the photocycle. (E) Proton pumping activity of heterologously expressed ActR-13023. (F) Illustration of naturally functional ActR-13023 as a light-driven outward proton pump.

Figure 4

Mutation studies of ActR-13023, wild-type ActR-13023, D85N, and E96Q mutants. (A) UV-visible spectra. (B) Light-induced differential spectra. (C) Proton pumping activities. pH titration and pKa calculation: (D) ActR-13023 spectra at pH 4, 7, and 10, (E) pKa value of ActR-13023, (F) pKa value of the D85N mutant, and (G) pKa value of the E96Q mutant.

Figure 5

Interaction between ActR-13023 and actinobacterial carotenoid. (A) Isothermal titration calorimetry (ITC) of ActR-13023 and the carotenoid. (B) UV-Vis spectrum of ActR-13023 after ITC experiment; the samples were shown in e-tube (1) purifiedActR-13023, (2) ActR-13023 after ITC experiment with carotenoid, and (3) the carotenoid as the ligand. (C) Comparison of proton pumping in the presence, absence of the carotenoid, and ActR-13023 treated with carbonyl cyanide m-chlorophenyl hydrazine (+CCCP).